Oxidative stress has been recognized as a main cause
of male subfertility with sperm DNA damage affecting
fertilization rates, embryo quality and pregnancy rates
within assisted reproductive technology (ART) cycles (
Under physiologic conditions, the oxy-redox balance,
besides leveraging on the assumption of reducing
substances with diet, is largely based on the endogenous
production of reducing power in the form of glutathione
(GSH), a reducing tripeptide with an activated sulfhydryl
group (SH) able to donate reducing equivalents and
reactivate most of the physiologic antioxidants (
Concept model of micronutrients administration in support to DNA methylation (blue pathway) and antioxidant defences (grey pathway). Micronutrients: small amounts of folates, vitamins B2, B3 and B12 and zinc are needed daily to feed the carbon cycle and the methylations (SAMe). The same applies to vitamin B6, zinc and cysteine to feed glutathione (GSH) synthesis. Cross activation: SAMe acts on the enzyme CBS to increase GSH synthesis. Reducing power from GSH synthesis in turn activates the carbon cycle. Homeostatic effects: DNA methylation and antioxidant defences synergize in keeping a healthy DNA status.
The above substances administered to male partners of
couples resistant to ART due to a male factor, resulted,
differently from what seen with strong oral antioxidants (
This experimental clinical trial study was approved by the Ethical Committee of Royan Institute (IR.ACECR. Royan.REC.1394.9) and carried out between April 2015 and April 2018 at the Isfahan Fertility and Infertility Center, Iran.
We included couples with male factor infertility, at least
1 failed ART cycle [either intra uterine insemination (IUI),
The male partners of the enrolled couples were prescribed with a 4-month treatment with a nutritional supplement containing micronutrients in support to the carbon cycle: folic acid (800 μg), vitamins B2 (2.8 mg), B3 (32 mg), B6 (2.8 mg) and B12 (5 μg), zinc (25 mg) and N-acetyl cysteine (500 mg) per day. Sperm quality was assessed at baseline and the end of the treatment.
The couples were followed-up for 3 to 10 months from treatment termination and pregnancies, either spontaneous or following a new ICSI cycle, were recorded.
The semen sample was collected by masturbation
after 3-7 days of abstinence before and after the
supplementation. Sperm parameters were assessed
according to the WHO (2010) criteria and motility was
assessed by CASA (CASA, Video Test, ltd: version
Sperm 2.1© 1990-2004, Russia). DNA fragmentation
was assessed by TUNEL (Apoptosis Detection System
Fluorescein, Promega, Germany) as previously reported
In case of couples undergoing a new ART cycle posttreatment, ovulation induction in the female partner was achieved by administration of recombinant follicle stimulating hormone (FSH, Sinal F, SinaGene, Iran) and human menopausal gonadotropin (hMG, Menogon, Ferring, Germany) after pituitary suppression by a gonadotropin release hormone (GnRH) antagonist (Cetrotide, Merk-Serono, USA). Follicular maturation was monitored by trans-vaginal ultrasounds and final ovulation was induced by 10000 IU human corionic gonadotropin (hCG, Choragon, Ferring, Germany). Oocytes were collected using ultrasound-guided trans vaginal aspiration and cumulus and coronal cells were removed to evaluate oocyte maturity. MII oocytes were selected for the following ICSI procedure.
Partner’s sperm was prepared for ICSI by density
gradient centrifugation (
Fertilization, cleavage, implantation, clinical pregnancy
and abortion rate were defined and assessed based on the
terminology of the international committee for monitoring
assisted reproductive technologies (ICMART) (
Statistical Package for the Social Sciences software (SPSS 18, Chicago, IL, USA) was used for data analysis. Data are expressed as mean ± error of the mean (SEM) and differences were considered significant at P<0.05. Comparison of sperm parameters, chromatin status, and lipid peroxidation, before and after treatment and clinical outcomes (fertilization, cleavage rate, top quality embryos) between current cycle and previous cycle was performed by Student’s t test. For comparison of pregnancy rate, Chi-square was used.
In total, 51 patients were enrolled and 8 of them quit for personal reasons. Data on semen analysis preand post-treatment were available for all 43 patients completing the study whereas some data concerning TUNEL (n=36), BODIPY, CMA3 and blue aniline (n=25) methods, were missing.
The supplementation with micronutrients had no
effect on sperm concentration, motility and volume
but significantly improved sperm morphology (n=43,
Sperm concentration (P=0.8), total motility (P=0.4), normal morphology (P=0.001), and volume (P=0.06) before and after a 4 month exposure to micronutrients, mean values ± SE.
Sperm protamine deficiency (P<0.05) and nuclear maturation (P<0.001) before and after a 4 month exposure to micronutrients, mean values ± SE (n=24). CMA3 reports on sperm protamine deficiency, Aniline blue reports on sperm nuclear maturation.
Sperm DNA fragmentation (P=0.001) and lipid peroxidation (P<0.001) before and after a 4 month exposure to micronutrients, mean values ± SE (n=24). TUNEL reports on sperm DNA fragmentation, BODIPY reports on lipid peroxidation.
Outcomes from the ART cycles and couples disposition. Fertilization, cleavage (P<0.05) and top quality embryo rates were compared to those achieved by the same couples in their previous ICSI cycle. ART; Assisted reproductive technology and ICSI; Intracytoplasmic sperm injection
Out of the 43 couples whose male partner completed the treatment, 25 couples did not undergo a new ART cycle and opted for an expectant management strategy. Four of these couples, achieved a spontaneous pregnancy during the study follow-up period resulting in 2 deliveries and 2 spontaneous miscarriages.
The remaining 18 couples underwent a new ICSI cycle but only 12 of them had embryos suitable for transfer or vitrification. Four of them underwent a fresh transfer cycle resulting in 3 clinical pregnancies and 3 abortions at either 3 weeks (n=1) or 2 months (n=2). Only 6 of 8 couples with vitrified embryos, underwent a thawed embryo transfer during the study period resulting in 3 clinical pregnancies with 2 deliveries and one early termination due to aneuploidy. The embryo cleavage rate was significantly higher (P<0.05) compared to the one in the previous cycle in the same couples whereas the improvement of fertilization rate and top quality embryo rate was not significant. The couples’ disposition and outcomes after a new ICSI cycle are summarized in Figure 5. Overall, treatment of 43 male partners of ARTresistant couples with micronutrients in support to the carbon cycle, resulted in 10 clinical pregnancies (23.3%) and 4 live births (9.3%).
This was a small size explorative study aimed at testing the hypothesis that a nutritional intervention using micronutrients in support to the carbon cycle, is of benefit to infertile men attending ART programs and that it is able to induce both improved DNA methylation and resumption of the endogenous antioxidant activity.
The administration of micronutrients did not
modify sperm count or motility but improved sperm
morphology, which can be assumed as a possible effect
on the processes of DNA methylation that contribute
to the epigenetic programming of the cell phenotype.
The parallel improvement of sperm nuclear maturation
shown by CMA3 and blue aniline staining, also points
to a possible effect on DNA methylation. Indeed, DNA
and histone methylation plays a pivotal role in sperm
nuclear maturation and is also involved in the process of
Micronutrients supplementation achieved a significant
reduction of sperm DNA fragmentation and lipid
peroxidation. The improvement of DNA fragmentation
was likely of clinical relevance because the average rate
moved from 23.2 to 17.8% (i.e. it dropped below the
critical threshold of 20% that is assumed as clinically
Micronutrients also achieved a significant drop in
sperm lipid peroxidation to half of the baseline value.
Lipid peroxidation is a process where oxidants attack
lipids containing carbon-carbon double bond(s),
especially polyunsaturated fatty acids (PUFAs), resulting
in production of other radicals attacking other double
bonds in a self-amplified process and is a clear-cut
oxidative damage (
In addition, a synergy with other micronutrients is likely
to apply. Abundance of SAMe from the carbon cycle can
bind CBS resulting in an allosteric activation with a fivefold
increase of enzyme activity (
Our study was too small in size to provide meaningful clinical findings; however, the outcomes were aligned with a possible positive effect of the treatment on the patients reproductive performance. Four out of 25 (16%) couples opting for an expectant management strategy, achieved a clinical pregnancy during the follow-up period. Interestingly, the male partners of these couples also showed a good response to the micronutrients of the sperm damage indexes (data not shown), which endorses a possible link between the positive pregnancy outcome and the treatment. Out of 18 couples opting for a new ART cycle, only 12 had viable embryos and only 10 of them underwent either fresh (n=4) or thawed (n=6) embryo transfer, which resulted in 6 clinical pregnancies. Again, the male partners of the couples achieving a pregnancy were good responders for sperm parameters. Altogether, these clinical outcomes indicate good chances that the treatment can be of help to male reproductive function at least in good responders. Reasons for lack of response may be bad treatment compliance, a negative genetic background and occurrence of other pathogenic mechanisms beside oxidative aggression, but our data do not allow to further speculate on this.
In summary, the oral administration of a combination
of micronutrients including folates, B vitamins, zinc
and cysteines to male partners of ART-resistant couples,
showed the ability to reduce sperm DNA damage and
improve sperm nuclear maturation and the clinical
outcomes reflected a positive effect. These outcomes were
related to the ability of the micronutrients to activate the
one carbon cycle resulting in both stronger methylation
ability and activation of the endogenous antioxidant
defences. The recorded antioxidant effect, which was
strong and likely of clinical value, was achieved without
perturbations of the cell homeostasis as happen with oral
The present study failed to address a series of relevant questions including the actual clinical gain that can be achieved by micronutrients, how to individuate potential good responders, dose and duration of the treatment and whether the add-on of oral antioxidants may further improve the effect or rather just derange the homeostatic regulations. All of these questions should be addressed by larger size clinical trials centred on the clinical outcomes. Meantime, micronutrients qualify as an alternative to antioxidants in correcting oxidative damage in infertile men.